ETS1 is a member of the ets gene family which includes ETS1 and ETS2 (Watson, et al., ERG, ELK1 and ELK2, SPil/PU.1, FLil, GABP.alpha., SAP1, ELF1, PEA3 and Drosophila melanogaster E47. The ETS1 gene is expressed in lymphoid cells, endothelial cells, astrocytes, but not detected in normal epithelial cells. It has previously been shown that the ETS1 gene is developmentally regulated in the mouse thymus. The ETS1 gene products are expressed at high levels in resting T-cells and its expression decreases following T-cell activation. The ETS1 gene encodes a nuclear protein and is phosphorylated by Ca.sup.2+ mediated events. It has been shown that the ets proteins bind to purine rich sequences (`GGA` core) in the promoter and enhancer region of cellular and viral genes and function as transcription factors. Among ETS family genes identified so far, only ETS1 and ETS2 have calmodulin-dependent kinase II consensus sequences, and have been shown to be phosphorylated by Ca.sup.2+ mediated events. Thus, the ETS1 protein may be a mediator of Ca.sup.2+ signals (generated by coupling of ligand to their cell surface receptors) into genetic reprogramming of specific set of genes involved in cellular function.
Two major isoforms of human ETS1 (i.e., p51 and p42) proteins have been identified in lymphoid cells and cell lines. p51 is encoded by full length ETS1 mRNA, while p42 is a product of alternatively spliced mRNA lacking exon VII. It has been shown that the major site of phosphorylation is in the sequences encoded by exon VII of ETS1, suggesting that p51, but not p42, may be involved in the Ca.sup.2+ mediated signal transduction processes. Also, it has been shown that p42 ETS1 or truncated form of ETS1 binds to DNA more efficiently than the full-length ETS1 protein. Several mechanisms including intramolecular repression, or the presence of an inhibitory domain have been proposed to explain weaker binding activities of the full-length ETS1 protein. However, it is possible that the p51, but not p42, may be able to interact with other proteins to form a complex that is not capable of binding to DNA. Alternatively, the flanking sequences outside the `GGA` core greatly influence the DNA-binding activities. The former hypothesis is strengthened by the recent demonstration that ETS1 family members are able to interact with other nuclear proteins. Sequence-specific binding of ELK1 is dependent on association with serum response factor; similarly associated GABP.alpha. with GABP.beta. and PU.1 with NFEMS enhances their capacity to bind DNA. However, an effort to demonstrate such a bonafide partners for ETS1 was not successful. Also, the temporal and tissue-specific expression pattern and different mode of regulation of ETS1 gene during T-cell activation suggest that ETS1 may be capable of either inducing or representing a certain set of genes. In lymphoid cells, identification of such ETS1-responsive genes is also complicated by the presence of multiple isoforms of ETS gene products lacking important functional domains, and therefore it is difficult to assign function to one or the other forms of ETS1.
To identify ETS1-responsive genes and to find out if the cellular phenotype plays any role in the ETS1 function, ETS1 has been expressed in DLD-1, a colon epithelial tumor cell line. The amounts of ETS1 expressed in transfected DLD-1 cells were comparable to those amounts expressed in lymphoid cells. The ectopically-expressed ETS1 has a relative molecular mass of 51 kDa and has an isoelectric point of 5.2. The ETS1 expressed in DLD-1 cells binds to the purine-rich motif containing 5'-CCGGAAGT-3', but not very well to the 5'-CAGGAAGT-3' (PEA3 motif), suggesting that the DNA-binding activity of ETS1 is influenced by flanking sequences outside the `GGA` core. It was also demonstrated that (i) the DNA-binding activity of ETS1 expressed in DLD-1 cells is very similar to ETS1 expressed in lymphoid cells and does not depend on cell background; (ii) the ETS1 produced in DLD-1 cells is biologically active and is capable of inducing a 54.5 kDa polypeptide; and (iii) the ETS1-responsive 54.5 kDa polypeptide is also expressed at high levels in lymphoid cells expressing abundant levels of ETS1 gene products.
Although it has been found that the tranfected DLD-1 cell line, i.e., a colon epithelial tumor cell line, exogenously expresses abundant levels of ETS1, the effect of ETS1 gene expression on cell growth and tumorigenicity has yet to be determined.